Tissue diversity is created through stem cell asymmetric self renewal division. Stem cell fate decision during division is thus fundamental for tissue homeostasis. Like other types of adult stem cells, hematopoietic stem cells (HSC) can produce daughter cells with distinct fates. Since both uncontrolled HSC expansion as well as loss of HSC is fatal for the organism, the decision of self renewal versus differentiation must be tightly controlled. An understanding of the molecular mechanism(s) of HSC fate decision is thus of considerable clinical importance. This application focuses on understanding the role of p190-B RhoGAP, a negative regulator of Rho GTPase activity, in HSC self renewal and fate decision. Our analysis of p190-B-null HSC shows that p190-B is essential for the regulation of HSC self renewal following transplantation. We show that loss of p190-B results in enhanced long term engraftment during serial transplantation. While the number of WT HSCs in the bone marrow of secondary recipients decreases in comparison with primary recipients, the numbers of p190-B-null HSCs is maintained between the secondary and primary recipients. At the same time, p190-B-null HSCs retain multipotential lineage differentiation. Thus, loss of p190-B prevents HSC exhaustion to maintain hematopoiesis during serial transplantation. At a mechanistic level, p190-B-deficiency did not alter HSC cell cycle kinetics or survival both in vivo and in vitro - thus, suggesting that p190-B modulates HSC self renewal activity as the cells divide. These data suggest the hypothesis that p190-B is a critical regulator of HSC self renewal by modulating HSC fate decision during divisions. We propose to identify the signaling pathway modulated by p190-B in HSC self renewal;and to determine the mechanism(s) by which p190-B and the associated signaling pathway regulates HSC fate decision. We plan to use functional assays of HSC functions, video microcopy, immunofluorescence imaging to examine (Aim 1) the role of IGF-1 signaling and p16Ink4a in p190-B-mediated HSC self renewal;(Aim 2) determine the role of p190-B and downstream signaling pathway identified in aim1 in mechanism of cell polarity that influences the segregation of cell fate determinant to control the balance of asymmetric/symmetric self renewal divisions. We believe that analysis of p190-B-null HSC offers a rare opportunity to identify stem cell-specific genes that are important for HSC fate decision. If our hypothesis that p190-B regulates HSC fate decision via IGF-1/p16Ink4a is validated, this will significantly change the view of IGF-1 signaling and p16Ink4a functions in HSC self renewal. The tremendous potential of HSC to reconstitute the hematopoietic system has allowed the development of clinical HSC transplantation to treat a wide variety of diseases. The proposed studies are expected to provide rationales to devise new therapeutic approaches of clinical HSC transplantation protocols. PUBLIC HEALTH RELEVANCE: Hematopoietic stem cell self renewal is a fundamental process to maintain continuous blood cell production throughout life. Deregulation of HSC self renewal leads to cancer or tissue degeneration. The tremendous potential of HSC to reconstitute the hematopoietic system has allowed the development of clinical HSC transplantation to treat a wide variety of diseases, including bone marrow failure or leukemias. The success of HSC transplantation is however limited due to our inability to control HSC functions. The studies proposed in this application will provide important information of the physiologic role of p190-B RhoGAP in the regulation of HSC self renewal. Therefore, our study will guide designing strategies to modulate HSC functions via inhibition of p190-B Rho GAP that could be used in the future to device new therapeutic approaches of clinical HSC transplantation protocols.